Part:BBa_K300002:Experience
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Applications of BBa_K300002
This part was tested through BBa_K300086.
Methods
Inoculum (into 5 ml LB+Amp) from glycerol stock of:
- BBa_K300086
- BBa_K300088
- BBa_K300090
- BBa_K300099
- BBa_K173000 (positive control)
- BBa_B0031 (negative control)
They were let grow ON at +37°C, 220 rpm.
The following day cultures were diluted 1:100 and let grow again for about five hours at +37°C, 220 rpm.
Optical density (O.D.) of each cell culture was than measured with TECAN Infinte F200. Samples were diluted in order to obtain the same O.D. equal to 0,02.
Than we performed a 21 hours' experiment with measurements of absorbance and green fluorescence every five minutes with TECAN Infinite F200. Each value shown is the mean of three measurements, from GFP data that of a non-fluorescent culture (negative control) was subtracted; cultures were shaken for 15 seconds every five minutes.
Results
 Mean (dGFP/dt)/O.D. over the exponential phase (under the hypothesis that GFP half-life in fusion contructs is similar to the original one -BBa_E0040) |
| Culture | Doubling time [min.] |
|---|---|
| BBa_K173000 | 77 |
| BBa_K300086 | 74 |
| BBa_K300088 | 75 |
| BBa_K300090 | 76 |
| BBa_K300099 | 76 |
| BBa_B0031 | 69 |
Discussion
All cell cultures showed a similar growth curve and doubling time was computed as described [http://2010.igem.org/Team:UNIPV-Pavia/Parts/Characterization#Doubling_time_evaluation here] in order to have informations about the burden due to synthesis of such fusion proteins. It is possible to see that all doubling time are very similar; it is possible to assert that the expression of these BioBrick parts does not cause abnormal stress to the cells.
In GFP curve it is possible to appreciate that in BBa_K300086, BBa_K300088, BBa_K300090, BBa_K300099 GFP accumulation it is very similar and it is significantly different from that of negative control BBa_B0031. These results show the right folding of the green fluorescent protein assembled downstream of the genetic circuit.
The mean protein synthesis rate was also computed over the growth exponential phase, showing again an appreciable GFP production rate that is about a half of the positive control.
User Reviews
UNIQ060aafb08c04a0e5-partinfo-00000013-QINU
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UNIPV-Pavia iGEM 2010 |
This part was used as a head domain alone and to construct the following synthetic composite affinity tags:
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UNIQ060aafb08c04a0e5-partinfo-0000001A-QINU